Progress 02/01/03 to 03/31/09

Outputs
OUTPUTS: Prolactinomas are tumors of the prolactin-secreting lactotropes in the pituitary gland. Prolactinoma development has been linked to estrogen exposure in both humans and animals. There are now several reports showing evidence for the existence of high levels of prolactin in chronic alcoholic men and women. We studied the cell signaling mechanism that mediates estradiol and ethanol actions on the cell to cell communication controlling lactotropic cell proliferation. Previously, we have shown that, like estradiol, ethanol mitogenic action on lactotropes depends on the communication between lactotropes and folliculostellate (FS). Recently, we reported that ethanol, like estradiol, reduces dopamine's ability to inhibit PRL release by modifying alternative splicing of the dopamine D2 receptor (Oomizu et al., 2003). We have also provided preliminary data showing that a TGF-beta1 system may mediate, in part, the growth-inhibitory action of dopamine on lactotropes. These data are now published as a feature article in Endocrinology. During the past funding period, we showed that ethanol increased basal and estradiol-enhanced wet weight and the prolactin content in the pituitary in a time-dependent manner. Chronic exposure of estradiol increased the levels of Gs protein in the pituitary. Unlike estradiol, ethanol exposure did not show significant effect on the basal level of Gs protein, but moderately increased the estradiol-induced levels of this protein. Estradiol exposure enhanced Gq11 protein levels in the pituitary after two and four weeks, while ethanol treatment failed to alter these protein levels in the pituitary in control-treated or estradiol-treated ovariectomized rats. In the case of Gi1, estradiol but not ethanol increased the level of this protein at four weeks of treatment. However, estradiol and ethanol alone reduced the levels of both Gi2 and Gi3 proteins at two and four weeks of treatment. Ethanol also significantly reduced the estradiol-induced Gi2 levels at four weeks and Gi3 level at two and four weeks. These results confirm ethanol's and estradiol's growth-promoting and prolactin stimulating actions on lactotropes of the pituitary and further provide evidence that ethanol and estradiol may control lactotropic cell functions by altering expression of specific group of G proteins in the pituitary. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our recent studies identified the cellular mechanism by which ethanol increases bFGF release from FS cells. We found that ethanol interact with TGF-beta3 and activate PKC- Ras-dependent MAPK p44/42 pathway to increase bFGF release from FS cells. We also found that TGF-beta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion. We have also begun to understand the role of dopamine in ethanol action on lactotropic cells proliferation. We identified that both ethanol and estradiol act on the dopamine receptor to alter D2 receptor-specific G proteins to activate the cAMP production which regulate both PRL production and cell proliferation in lactotropes of the pituitary gland.

Publications

• No publications reported this period

Progress 01/01/07 to 12/31/07

Outputs
Prolactinomas are tumors of the prolactin-secreting lactotropes in the pituitary gland. Prolactinoma development has been linked to estradiol and alcohol exposure in both humans and animals. During the last year, we determined whether ethanol's and estradiol's lactotropic cell-proliferating actions are associated with alteration in the production of three peptides that regulate cell growth: transforming growth factor beta 1 (TGF-beta1), TGF-beta3 and basic fibroblast growth factor (bFGF). Using ovariectomized Fischer-344 female rats, we determined ethanol's and estradiol's actions on lactotropic cell proliferation and growth-regulatory peptide production and release in the pituitary gland during tumorigenesis. Ethanol increased basal and estradiol-enhanced mitosis of lactotropes in the pituitary glands of ovariectomized rats. The level of growth-inhibitory TGF-beta1 was reduced in the pituitary following ethanol and/or estradiol treatment for 2 and 4 weeks. In contrast, ethanol and estradiol alone as well as together increased levels of growth-stimulatory TGF-beta3 and bFGF in the pituitary at 2 and 4 weeks. In primary cultures of pituitary cells, both ethanol and estradiol reduced TGF-beta1 release and increased TGF-beta3 and bFGF release at 24 h. Ethanol's effect on growth factor levels in the pituitary or growth factor release from the pituitary cells was less than that of estradiol. When ethanol and estradiol were applied together, their individual effects on these growth factors were amplified. These results provide evidence that both estradiol and ethanol alter the production and secretion of growth-regulatory peptides controlling lactotropic cell proliferation. We have also determined the changes in the pituitary levels of G proteins during the tumor development following alcohol and ethanol treatments. We found that ethanol increased basal and estradiol-enhanced wet weight and the prolactin content in the pituitary in a time-dependent manner. Chronic exposure of estradiol increased the levels of Gs protein in the pituitary. Unlike estradiol, ethanol exposure did not show significant effect on the basal level of Gs protein, but moderately increased the estradiol-induced levels of this protein. Estradiol exposure enhanced Gq11 protein levels in the pituitary after 2 and 4 weeks, while ethanol treatment failed to alter these protein levels in the pituitary in control-treated or estradiol-treated ovariectomized rats. In the case of Gi1, estradiol but not ethanol increased the level of this protein at four weeks of treatment. However, estradiol and ethanol alone reduced the levels of both Gi2 and Gi3 proteins at two and four weeks of treatment. Ethanol also significantly reduced the estradiol-induced Gi2 levels at four weeks and Gi3 level at 2 and 4 weeks. These results provide evidence that ethanol and estradiol control lactotropic cell functions by altering the expression of specific group of G proteins in the pituitary.

Impacts
Our recent studies identified the cellular mechanism by which ethanol increases lactotropic cell proliferation. Ethanol, like estradiol, alters the production and secretion of growth-regulatory peptides controlling lactotropic cell proliferation. Additionally, we began to unfold cellular mechanism of both estradiol and ethanol actions on lactotroes. Our data provide evidence that both ethanol and estradiol control lactotropic cell functions by altering expression of specific group of G proteins in the pituitary.

Publications

• 1. Sarkar DK, Boyadjieva N. 2007 Ethanol alters production and secretion of estrogen-regulated growth factors that control prolactin-secreting tumors in the pituitary. Alcohol Clin Exp Res. 31:2101-2105.
• 2. Chaturvedi K, Sarkar DK 2007 Alteration in G proteins and prolactin levels in pituitary after ethanol and estrogen treatment. Alcohol Clin Exp Res. In Press

Progress 01/01/06 to 12/31/06

Outputs
Prolactinomas are tumors of the prolactin-secreting lactotropes in the pituitary gland. Prolactinoma development has been linked to estrogen exposure in both humans and animals. There are now several reports showing evidence for the existence of high levels of prolactin in chronic alcoholic men and women. We studied the cell signaling mechanism that mediates estradiol and ethanol actions on lactotropic cell proliferation and prolactin release. Recently we have shown that both estrogen and ethanol down-regulate dopamine receptor short form (D2S) in the pituitary. Since dopamine receptor is G protein coupled receptor, the possibility arises that the down stream action of ethanol may involve alteration in the levels of G proteins. The members of Gi subfamily including Gi1, Gi2 and Gi3 generally transduce inhibitory signals by reducing adenylyl cyclase activity and intracellular calcium levels. We found that both ethanol and estradiol reduced the level of Gai2 and Gai3 proteins after 2-4 weeks of treatment. These data support that the mechanisms that mediate estradiol and ethanol actions on prolactin may involve suppression of inhibitory G proteins. Estradiol action on prolactin production may also involve the stimulatory G proteins. We also investigated whether ethanol and estradiol interact to modulate D2 receptor splicing to activate G-proteins in lactotropes. First, we determined the effects of ethanol and estradiol on G protein levels in three different types of lactotropic cells: one with knocked out D2S and D2L receptors called pcDNA, one expressing only D2S receptors, and one expressing D2L receptors. Bpth estradiol and ethanol produced a dose-dependent suppression of Gialpha protein level and increase of Gsalpha levels in the D2S cell line thus showing that these proteins are inhibited and activated respectively. In addition in the D2L cell line these agents resulted in an increase in both Gialpha and Gsalpha proteins thus determining that these proteins are also linked to this specific receptor. Moreover, Gialpha and Gsalpha protein levels within the pcDNA cell line did not change, concluding that D2 receptors are imperative for Gialpha3 and Gsalpha protein alteration. Because G proteins alter mitogen activated protein kinase p44/42 (MAPK) activity in lactotropes, we determined whether or not ethanol increased the phosphorylation of MAPK in pcDNA and D2S cells. Furthermore, the effect of MAPK inhibitor U0126 on ethanol-induced prolactin release was determined in these cells. Ethanol increased the release of prolactin from pcDNA cells. Ethanol failed to increase prolactin release from D2S or pcDNA cells, which suggests that a reduction in D2S receptor functions may be a mechanism by which ethanol alters prolactin secretion from lactotropes. Ethanol also increased MAPK phyosphorylation in pcDNA cells, while it did not activate MAPK in D2S cells. Additionally, MAPK inhibitor suppressed ethanol-induced prolactin release from pcDNA cells, which suggested involvement of MAPK pathway in ethanol action on prolactin secretion.

Impacts
The data obtained from our studies increased the understanding of the action of estradiol and ethanol on pituitary lactotropes, particularly prolactin secretion. We have shown that G protein levels within lactotropic cells may be altered through the introduction of estradiol or ethanol exposure. These studies also have identified that dopamine D2S receptors are obligatory for activating Gialpha3 proteins for prolactin production in lactotropes. These studies have demonstrated that the downstream signaling of ethanol action may involve activation of MAPK. Therefore, it can be hypothesized that using dopamine D2S receptors as a specific target for lactotropes inhibiting drugs may be a more effective way of treating the prolactinomas instead of using the traditional dopamine agonist.

Publications

• Sarkar DK. 2006 Genesis of prolactinomas: studies using estrogen-treated animals. Front Horm Res. 35:32-49.

Progress 01/01/05 to 12/31/05

Outputs
Prolactinomas are tumors of the prolactin-secreting lactotropes in the pituitary gland. Prolactinoma development has been linked to estrogen exposure in both humans and animals. There are now several reports showing evidence for the existence of high levels of prolactin in chronic alcoholic men and women. We studied the cell signaling mechanism that mediates estradiol and ethanol actions on the cell to cell communication controlling lactotropic cell proliferation. Previously, we have shown that, like estradiol, ethanol mitogenic action on lactotropes depends on the communication between lactotropes and folliculostellate (FS). Our recent study identified that the PKC-activated Ras-dependent mitogen activated protein kinase (MAPK) p44/42 pathway is involved in the cross-talk between transforming growth factor (TGF)-beta3 and ethanol to increase bFGF release from FS cells (Publication 1). FS cells are known to communicate with each other and with endocrine cells via gap-junctions in the anterior pituitary. We investigated whether TGF-beta3 and estradiol, known to regulate FS cell production and secretion of bFGF, increases gap-junctional communication to alter bFGF secretion from FS cells. FS cells in monolayer cultures were treated with TGF-beta3 or vehicle alone for 24 h and then microinjected with Lucifer Yellow and high molecular weight Texas Red dextran. Ten minutes later, the transfer of dye among adjacent cells was recorded with a digital microscope. TGF-beta3 increased the transfer of dye. The TGF-beta3 neutralizing antibody and the gap-junction inhibitor octanol reduced the effect of TGF-beta3 on the transfer of dye. The TGF-beta3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGF-beta3 increased total and phosphorylated levels of connexin 43 (Cx43). Estradiol treatment did not produce any significant changes on basal or TGF-beta3 induced increases in Cx43 levels. The gap-junction inhibitor octanol reduced TGF-beta3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGF-beta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion (Publication 2). During the past funding period, we reported that ethanol, like estradiol, reduces dopamine ability to inhibit PRL release by modifying alternative splicing of the dopamine D2 receptor (Oomizu et al., 2003). We also provided preliminary data showing that a TGF-beta1 system may mediate, in part, the growth-inhibitory action of dopamine on lactotropes. These data are now published as a feature article in Endocrinology (Publication 3).

Impacts
Our recent studies identified the cellular mechanism by which ethanol increases bFGF release from FS cells. We found that ethanol interact with TGF-beta3 and activate PKC- Ras-dependent MAPK p44/42 pathway to increase bFGF release from FS cells. We also found that TGF-beta3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion. We have also begun to understand the role of dopamine in ethanol action on lactotropic cells proliferation.

Publications

• 2. Kabir N, Chaturvedi C, Liu LS, Sarkar DK. 2005 TGF-beta3 increases gap junctional communication among folliculostellate cells to increase bFGF release. Endocrinology 146:4054-4060 [Cover page article].
• 3. Sarkar DK, Chaturvedi K, Oomizu, S, Boyadjieva N, Chen CP. 2005 Dopamine and TGF-beta1 interact to inhibit the growth of pituitary lactotropes. Endocrinology 146:4179-4188 [A feature article].
• 1. Chaturvedi K, Sarkar DK. 2005 Role of PKC-Ras-MAPK p44/42 in ethanol and TGF-beta3 induced bFGF release from folliculostellate cells. J Pharm Exp Ther 314:1346-1352.

Progress 01/01/04 to 12/31/04

Outputs
Prolactinomas are tumors of the prolactin-secreting lactotropes in the pituitary gland and are the most frequently occurring neoplasms in the human pituitary. In the general population, 1 in 2800 men and 1 in 1050 women have prolactinomas. Patients with prolactinomas typically show elevated levels of plasma prolactin (hyperprolactinemia). Prolactinoma development has been linked to estrogen exposure in both humans and animals. There are now several reports showing evidence for the existence of high levels of prolactin in chronic alcoholic men and women. We have recently obtained data showing that ethanol causes hyperprolactinemia by increasing prolactin release from lactotropes and by increasing lactotropic cell proliferation. Ethanol also potentiates estradiol-induced prolactinomas in ovariectomized female rats and induces prolactinomas in cyclic female rats. Furthermore, we have provided evidence that ethanol acts directly on lactotropes to increase prolactin secretion, whereas ethanol increases cell proliferation by altering cell-to-cell communication between pituitary cells. We are continuing to study the cellular mechanism of ethanol's action and comparing the ethanol and estradiol actions on lactotrope growth. Studies conducted during the past years have resulted in an increased understanding of the action of ethanol on pituitary lactotropes. We have previously shown that under ethanol's influence, lactotropes secrete transforming growth factor beta 3 (TGF-B3), which increases the release of basic fibroblast growth factor (bFGF) from FS cells to control lactotropic cell proliferation in the pituitary gland. Our recent studies identified the cellular mechanism by which bFGF increases cell proliferation. We found that bFGF-increased lactotropic cell proliferation is mediated by activation of Src kinase, Ras p21 and MAP kinase p44/42. We have also begun to understand the role of dopamine in ethanol's action on lactotropic cells proliferation. We have previously shown that ethanol, like estradiol, reduces dopamine's ability to inhibit PRL release by modifying alternative splicing of the dopamine D2 receptor in the pituitary. In this study, we found, for the first time, that dopamine inhibits lactotropic cell proliferation by increasing TGF-B1 production. These data provide the exciting possibility that like estradiol, ethanol increases lactotropic secretion of TGF-B3 that stimulates FS-cell release of bFGF which activates Src kinase, Ras p21 and MAP kinase p44/42 to enhance lactotropic cell proliferation. In addition, ethanol-increased lactotropic cell proliferation may also involve reduction in the dopamine-activated lactotropic cell growth-inhibitory TGF-B1 production due to altered D2-receptor splicing. Together these data not only provide information on the cellular actions of ethanol and estradiol on lactotropes but also indicate the possibility of a novel growth-promoting action of ethanol on the pituitary tissues and possibly on other tissues. This new information is significant in understanding the etiology and treatment of hyperprolactinemia.

Impacts
These data support the concept that the cell-cell communication between the lactotropes and follicular stellate cells is critical for the development of pituitary prolactin-secreting tumors following chronic alcohol consumption.

Publications

• No publications reported this period

Progress 01/01/03 to 12/31/03

Outputs
We are continuing to study the cellular mechanism of ethanol's action on lactotrope growth. Our results thus far suggest that chronic ethanol administration elevates plasma levels of prolactin both in the presence and absence of estradiol. Possibly ethanol increases plasma levels of prolactin by increasing the number of prolactin-secreting lactoropes, and by stimulating prolactin gene expression and secretion from lactotropes. Ethanol also potentiates estradiol-induced prolactin gene expression, prolactin secretion and lactotrope proliferation. Both ethanol and estradiol activate prolactin expression by acting directly on lactotropes and altering D2 and TRH-regulated mechanisms. Ethanol and estradiol also alter lactotrope proliferation, possibly by increasing cell-cell communication between lactotropes and FS cells by altering secretion of TGF-beta1, TGF-beta3 and bFGF. Furthermore, our studies have provided evidence to support a role of MAP kinase signaling in ethanol- and estradiol-action on growth factor production. Finally, these data suggest, for the first time, that ethanol and estradiol interact at the cellular level to control lactotropic cell function. These are major findings-they identify a cause for the increased incidence of breast and pituitary tumors in alcoholic patients. Together these data not only provide information on the cellular action of ethanol on lactotropes but also indicate the possibility of a novel growth-promoting action of ethanol on the pituitary tissues and possibly on other tissues. This new information is significant in understanding the etiology and treatment of hyperprolactinemia in alcoholics.

Impacts
These data provide the first evidence that chronic alcohol consumption promotes the development of pituitary tumors by acting like estrogen at the cellular level in lactotropes.

Publications

• Oomizu S, Boyadjieva N, Sarkar DK 2003 Ethanol and estradiol modulate alternative splicing of dopamine D2 receptor mRNA and abolish the inhibitory action of bromocriptine on prolactin release from the pituitary gland. Alcohol Clin Exp Res 27:975-80.
• Chaturvedi K, Sarkar DK. 2004 Involvement of PKC dependent p44/42 MAP kinase signaling pathway for cross-talk between estradiol and TGF-beta3 in increasing bFGF in folliculostellate cells. Endocrinology 145:706-15.
• Caporali S, Imai M, Altucci L, Cancemi M, Caristi S, Cicatiello L, Matarese F, Penta R, Sarkar DK, Bresciani F, Weisz A 2003 Distinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 Cells. Mol Bio Cell 14:5051-59.
• Goya RG, Sarkar DK, Brown OA, Herenu CB 2004 Potential of gene therapy for the treatment of pituitary tumors. Current Gene Therapy.(In press).
• Oomizu S, Chaturvedi K, Sarkar DK. 2004 Folliculostellate cells determine the susceptibility of lactotropes to estradiol's mitogenic action. Endocrinology. (In press).